HUMAN B-CELLS SECRETING IMMUNOGLOBULIN-G TO GLUTAMIC-ACID DECARBOXYLASE-65 FROM A NONDIABETIC PATIENT WITH MULTIPLE AUTOANTIBODIES AND GRAVES-DISEASE - A COMPARISON WITH THOSE PRESENT IN TYPE-1 DIABETES

Antibodies to glutamic acid decarboxylase-65 (GAD65) are present in a number of autoimmune disorders, such as insulin-dependent (type 1) dia betes mellitus (IDDM), stiff man syndrome, and polyendocrine autoimmun e disease. Antibodies to GAD in IDDM patients usually recognize confor mation-dependent regions on GAD65 and rarely bind to the second isofor m, glutamic acid decarboxylase-67 (GAD67). In contrast, those present in stiff man syndrome and polyendocrine disease commonly target the se cond isoform (GAD67) and include antibodies that are less dependent on the conformation of the molecule. By immortalizing peripheral blood B cells with Epstein-Barr virus, ne have generated three human IgG auto antibodies, termed b35, b78, and b96, to GAD65 from one patient with m ultiple autoantibodies to endocrine organs and Graves' disease. All th ree ee autoantibodies are of the IgG1 isotype, with islet cell activit y, and do not react with GAD67. The regions on GAD65 recognized by the three autoantibodies have been investigated by immunoprecipitation wi th a series of chimeras, by binding to denatured and reduced antigens, and using protein footprinting techniques. Using chimeric GAD protein s, we have shown that b35 targets the IDDM-E1 region of GAD65 (amino a cids 240-435) whereas both b78 and b96 target the IDDM-E2 legion of GA D65 (amino acids 451-570). Furthermore, examination of binding to reco mbinant GAD65 and GAD67 by Western blotting revealed some differences in epitope recognition, where only b78 bound denatured and reduced GAD 65. However, b35, b78, and b96 autoantibodies had different footprinti ng patterns after trypsin treatment of immune complexes with GAD65, ag ain indicating different epitope recognition. Our results indicate tha t antibodies to GAD65 present in nondiabetic patients with multiple au toantibodies to endocrine organs show similarities to those in IDDM (b y targeting IDDM-E1 and IDDM-E2 regions of GAD65) as well as subtle di fferences in epitope recognition (such as binding to denatured and red uced GAD65 and by protein footprinting). Thus, the GAD65 epitopes reco gnized by autoantibodies in different autoimmune diseases may overlap and be more heterogeneous than previously recognized.